(1) Field of the Invention
The present invention generally relates to recombinant mycobacteria. More specifically, the invention is directed to mycobacteria that elicit IL-12 production in infected macrophages.
(2) Description of the Related Art
Tuberculosis (TB) is currently the second leading cause of death from a single infectious disease worldwide (Frieden et al., 2003; Onyebujoh and Rook, 2004). It is well established that Mycobacterium tuberculosis possesses a variety of mechanisms that promote enable evasion of innate immunity and permit latent infection in the presence of host adaptive immune responses (Flynn and Chan, 2003; Glickman and Jacobs, 2001; Flynn and Chan, 2001). This latent reservoir of M. tuberculosis can eventually develop into active disease when an individual's immune system is compromised due to aging, chemotherapy, or infection with HIV (Stead and Dutt, 1991; Kim et al., 2005; Ito et al., 2005; Campos et al., 2003). Currently, the attenuated M. bovis strain BCG is the only vaccine available for routine human immunization. It has had little if any impact on the increasing global prevalence of TB, in spite of having been administered to more than a billion people (Behr and Small, 1997). Thus, work on developing new and more immunogenic vaccine candidates is crucial and requires advances in our understanding of the host-pathogen interaction.
Cytokines play an important role in controlling intracellular infection, and the cytokine profile produced within the first days or weeks following infection can define the type of host immunity induced and, thereby, the outcome of the disease. A critical cytokine in the control of intracellular infections is interleukin-12 (IL-12), which is produced mainly by macrophages and dendritic cells (DCs) (Manetti et al., 1993; Trinchieri, 2003). IL-12 activates both early nonspecific innate immune resistance and subsequent antigen-specific adaptive immune responses. IL-12 is a growth factor for activated NK cells and induces them to produce IFNγ, a potent activator of macrophage bactericidal activity, while also inducing their cytotoxic activity. IL-12 also stimulates naïve T-cells, a component of adaptive immunity, to differentiate into IFN-γ producing T helper type 1 (Th1) cells.
IL-12p70, a dimer of IL-12p35 and IL-12p40, was originally thought to be the only bioactive form of IL-12 that activates both NK and T-cells to produce IFN-γ. It is now clear that other forms of IL-12 also have important immunoregulatory functions. Dimerization of IL-12p40 with the p19 subunit forms IL-23, which has IFN-γ inducing activity. The IL-12p40 subunit can also homodimerize, and recent studies indicate that the IL-12p40 homodimer is required for initiating adaptive immunity before IL-12p70 or IL-23 are produced (Khader et al., 2006). Thus, full activation of Th1 immunity is regulated by multiple cytokines containing the IL-12p40 subunit. The expression of the IL-12p40 subunit is regulated in infected myeloid cells, such as macrophages (Trinchieri, 2003), and it is known that mice and humans with mutations in the IL-12p40 gene or its cognate receptor are highly susceptible to mycobacterial infection (Altare et al., 2001; Altare et al., 1998; Jouanguy et al., 1999; de Jong et al., 1998; Cooper et al., 2002; Cooper et al., 1997; Cooper et al., 1995). However, other IL-12p40-related cytokines, such as IL-23 and their receptors, may be dispensable, since the survival rate of mice lacking these cytokines is comparable to that in wildtype mice following M. tuberculosis infection (Khader et al., 2005).
In the mouse model of infection, M. tuberculosis persists in macrophages even though induction of Th1 immunity is observed around 4 weeks post-infection (Flynn, 2004). Although bacterial growth slows and bacterial load eventually reaches a stable plateau as a result of adaptive immunity, the pathogen is not eliminated. Moreover, bacterial burden remains 2 to 3 orders of magnitude above that of attenuated strains of M. tuberculosis, such as H37Ra (Jung et al., 2002). Among other complications, the high bacterial burden and progressive chronic inflammation associated with it contribute to the death of the animal. While an effective Th1-mediated immune response generally develops following infection with intracellular pathogens such as Salmonella or Toxoplasma gondii, M. tuberculosis appears to evade eradication, in part by preventing the development of an effective Th1 response (Jung et al., 2002; Mastroeni, 2002; Gazzinelli et al., 1994). Studies of the mechanisms of immune evasion by M. tuberculosis have revealed that one of the strategies used by the tubercle bacillus is to repress macrophage production of IL-12p40 (Hickman et al., 2002; Nau et al., 2002). This parallels the paradigm proposed for Leishmania infection, in which repression of IL-12 production has been established as a key immune evasion strategy of the parasite (Reiner et al., 1994; McDowell and Sacks, 1999). Taken together, these findings suggest that during M. tuberculosis infection the host mounts a limited Th1 immune response that is insufficient to completely eliminate the tubercle bacterium, because specific determinants produced by M. tuberculosis modulate the host immune response.
ESAT-6 gene clusters. The primary attenuating mutation of bacillus Calmette-Guerin (BCG) was found to be a deletion (namely RD1), which was shown to encode two secreted proteins and their specific secretion system. These secreted proteins, ESAT-6 and Cfp-10, were first identified in culture filtrates of M. tuberculosis and shown to be immunodominant antigens. More recently, these proteins have been demonstrated to mediate necrosis of an infected cell which leads to egress of M. tuberculosis from the infected cells. Interestingly, ESAT-6 and Cfp-10 are part of a family of related homologues within M. tuberculosis and related orthologues in mycobacterial species (Gey van Pittius et al. 2001). Moreover, this family of proteins are found in all gram-positive organisms and deletion in Staphylococcus causes attenuation.
Based on the above, it would be useful to identify mycobacterium genes that affect host IL-12 production and to further characterize ESAT-6 and ESAT-6-like gene clusters, particularly with regard to virulence. The present invention addresses that need.